Casein (CN) is the predominant protein in human and non-human mammal's milk. Casein has been characterized previously as composed of three fractions, α, β and γ, according to the electrophoretic mobility of each fraction (Hipp et al. 1952. Dairy Sci., 35:272). Today, casein is defined according to the amino acid sequences of each of the subgroups αS1, αS2, P and K (Engel et al. 1984. J. Dairy Sci. 67:1607-1608).
Enzymatic hydrolysis of casein liberates peptides that may contribute to the health and proper development of young (FitzGerald et al., 1998. Int. Dairy J. 8:451-457) and that serve as local regulators of mammary gland function (Silanikove et al., 2000. Life Sci. 67:2201-2212; Shamay et al., 2002. Life Sci. 70:2707-2719). The serine protease, plasmin, is the predominant protease in milk and is known to produce boiling-resistant peptides (proteose-peptones) from β-casein, α-S1 casein and αS2-casein.
The Proteose-peptones (PPs), also known as casein phosphopeptides (CPP), constitute about a third of the whey proteins (Andrews, 1983. J. Dairy Res. 50:45-55). Plasmin in milk is found mainly in its inactive from, plasminogen, and the conversion of plasminogen to plasmin is modulated by plasminogen activators (Politis I., 1996. J. Dairy Sci. 79:1097-1107).
Casein-derived peptides have been shown to have several biological activities and applications. Studies with milk compounds demonstrated casein-related bactericidal activity. U.S. Pat. No. 3,764,670 discloses novel polypeptides derived from casein possessing antibiotic properties against microorganisms.
Immune modulating activity of casein peptides has also been observed. For example, International PCT Patent Application WO 01/13739 discloses method of potentiating the immunity of mammals and promoting the growth thereof by administering proteins containing phosphorylated amino acids such as casein phosphopeptides, directly or in food. As the immunity of the mammals is potentiated, the resistance thereof against infectious diseases is strengthened and factors inhibiting the growth thereof are eliminated, thereby promoting the growth of the mammals.
U.S. Patent Applications Publication Nos. 20020147144 and 20040167073 disclose biologically active peptides that are derived from or are similar to sequences identical to the N-terminus of the αS1 fraction of milk casein, which are capable of stimulating and enhancing immune response, protecting against viral infection, normalizing serum cholesterol levels, and stimulating hematopoiesis.
International PCT Patent Application WO 2005/081628 discloses biologically active peptides that are derived from or are similar to sequences of the αS1, αS2, β- or κ-casein fractions of milk casein, capable of immune modulation and other therapeutic activities, including but not limited to stimulating and enhancing immune response, protecting against viral infection, normalizing serum cholesterol levels, and stimulating hematopoiesis. The casein-derived peptides are non-toxic and can be used to treat and prevent immune pathologies, diabetes, hypercholesterolemia, hematological disorders and viral-related diseases.
European Patent Application No. EP1375513 discloses that among the peptides derived from casein, peptides having amino acid sequences comprising plural phosphoserine residues show a strong immuno-enhancing activity. Specifically, the invention relates to an immuno-enhancing agent comprising a peptide consisting of the amino acid sequence Q1-SerP-X-SerP-Q2, wherein, SerP represents the phosphoserine residue, X represents one to three of any amino acid residues, and Q1 and Q2 are independently absent or represents at least one of any amino acid residue.
U.S. Pat. No. 6,391,849 to one of the inventors of the present invention and co-workers discloses casein-derived proteose-peptones that act as calcium chelators, and their use in controlling physiological changes in a mammary gland, including transient and persistent cessation of milk production, induction of involution and prevention, treatment and reversal of infections.
Casein phosphopeptides have been shown to possess the unique property of being able to bind macroelements such as Ca, Mg, and Fe, along with trace elements such as Zn, Ba, Cr, Ni, Co and Se, which may be solubilized in the small intestine and therefore available for absorption. As such, CPPs are used as additives in beverages and infant food, and in dental medicaments. For example, U.S. Pat. No. 5,834,427 discloses a purified casein phosphopeptide (CPP) having a novel amino acid sequence and purified casein including same. The CPP or the casein containing same has an improved ability of solubilizing minerals and absorbing thereof in animals. The CPP or the β-casein-H containing same can be added to foodstuffs, beverages, medication, cosmetics, feed in an effective amount of enhancing a mineral absorption in animals. An oral composition comprising the beta-casein H or the inventive CPP and a pharmaceutically acceptable carrier can reduce or relieve a dentinal hypersensitivity.
U.S. Pat. No. 5,227,154 discloses a method of controlling dental calculus by treating the teeth with an oral composition which comprises specific casein phosphopeptides and/or salts thereof. U.S. Pat. No. 6,652,875 discloses a formulation for the delivery of bioactive constituents to biological surfaces, including dental surfaces such as teeth and gums, wherein said formulation comprises a suspensions or solution of at least one isolated and purified casein protein or salt thereof, in water, together with at least one bioactive constituent.
Various methods for the preparation of casein hydrolyzates, specifically CPPs, have been proposed. For example, U.S. Pat. No. 4,740,462 discloses production of CPP by hydrolysis of casein with a crystalline trypsin followed by fractionation and separation by ultrafiltration or chromatographic techniques such as gel permeation chromatography or ion-exchange chromatography. This method may have some research utility, but it is not suitable or economical on an industrial scale. Other methods involve the use of toxic substances, such as barium chloride, which are not acceptable in food product and/or pharmaceutical compositions.
Regardless of the method used for preparation, a solution containing a casein hydrolyzate tends to be turbid. Turbidity is considered as a significant disadvantage in pharmaceutical compositions as well as in some food products, specifically beverages, as it is difficult or impossible to visibly track changes in the turbid composition, particularly to detect contaminations. U.S. Pat. No. 5,405,756 discloses a method for the preparation of casein phosphopeptide suitable for use as an additive to beverages without affecting the beverage transparency; however, the obtained protein contains calcium, and, moreover, a clear solution is obtained only at an acidic pH.
Management of Lactating Livestock Animals
In the modern dairy industry, lactating animals in herds go through controlled cycles of milking and pregnancy, as such regimes contribute to a significant increase in milk production. In current management of dairy herds, for example cows and goats, there is a significant overlap between lactation and pregnancy, wherein a “dry period” is imposed between 50 to 70 days prior to parturition by cessation of milking. This regime is set to compromise between the need to induce involution, a necessary process for subsequent healthy lactating period, and the requirement for high milk production all year long.
Cessation of milk removal leads to rapid changes in the mammary secretion and to initiation of the process of active mammary involution. This process comprises an extensive and highly ordered sequence of changes in tissue and milk composition, which occur during the transition between the lactating and the non-lactating states. During the first stage of mammary involution, the process is triggered by local stimuli that initiate apoptosis, but involution can be reversed by reinitiating milk removal (Capuco and Akers, 1999. J. Mammary Gland Biol. Neoplasia 4:137-144; Wilde et al., 1999. J. Mammary Gland Biol. Neoplasia 4:129-136). This local control can cause involution when milk stasis is induced in individual glands, as was observed in lactating goats following unilateral cessation of milking (Quarrie et al., 1994. Biochem. Soc. Trans. 22:178S), or in lactating mice following teat sealing (Li et al., 1997. Proc. Natl. Acad. Sci. U.S.A. 94: 3425-3430; Marti et al., 1997. Eur. J. Cell. Biol. 73:158-165).
The second stage of involution is persistent, and milk removal cannot cause resumption of milk secretion (Capuco and Akers, 1999. ibid; Wilde et al., 1999. ibid). Reversal of the second state of involution can occur only in a subsequent lactating stage after giving birth. This stage is characterized by activation of proteases that destroy the lobular-alveolar structure of the gland by degrading the extracellular matrix and basement membrane, as well as massive loss of alveolar cells.
The cessation of milking to induce involution is associated with increased risk of developing mastitis, a disease caused by intramammary infection (IMI) with pathogens, mostly bacteria, but also yeast, fungi, or even algae. Mastitis can be clinical, with local (and in some cases general) clinical signs and milk abnormalities, or subclinical with production losses and lowered milk quality.
Modern dairy cows are usually dried while still producing 20 to 40 liters of milk per day. Therefore, milk stasis may cause leaking of mammary secretion, which substantially increases the risk of acquiring IMI. Conventional dry-off, leading to a long process of involution, is typically associated with a higher rate of IMI. Clinical and subclinical mastitis produce significant economic losses due to rejected milk (less farm production), degraded milk quality (less revenue), early culling of cows (loss of genetic potential), drug costs, veterinary expenses, and increased labor costs for the farmer. Mastitis is the most debilitating disease in dairy herds, costing in the U.S. dairy industry alone about $2 billion annually.
It has previously been shown by one of the inventors of the present invention and co-workers that a pure β-casein (β-CN) fraction 1-28 down-regulates milk secretion in cows and goats. The activity of this peptide was correlated with its ability to block potassium channels in the apical membranes of mammary epithelia (Silanikove et al., 2000. supra).
It was also shown that injection of crude preparation of casein hydrolyzates (CNH) into the udder of a goat or a cow mimics the natural phenomenon of involution, inducing a local inflammatory response and loss of tight junction (TJ) integrity, followed by rapid drying-off of mammary secretion (U.S. Pat. No. 6,391,849; Shamay et al., 2002. ibid; Shamay et al., 2003. J. Dairy Sci. 86:1250-1258). The process induced by CNH was more rapid and synchronized than that induced at natural drying-off. These results indicate that it is possible to significantly reduce the time required for involution. However, it is still unclear whether it is possible to shorten or omit the dry period without affecting the milk yield in the subsequent lactation period. Annen et al. (2004. J Dairy Science 87:3746-3761), showed that treatment of multiparous cows with bovine somatotropin (bST) enabled shortening, and even omitting, the dry period without reduction in milk production; however, this treatment was not as effective for primiparous cows. Moreover, it has been previously shown that treatment with bST causes mastitis, reproductive disorders and other production related diseases, and that such treatment increases foot disorders.
Farm animal welfare is of increasing public concern in Western societies in the last decades (Broom D M 1992 In: Phillips et al., Eds. Farm Animals and the Environment. CAB Wallingford UK pp 245-253). Recent development in housing and management practices of farm animals under intensive production systems reflects the increase in moral concerns of animal welfare (Fregonesi et al., 2001. Livestock Production Sci. 68:205-216; Fregonesi et al., 2002. Livestock Production Sci. 78:245-257). Improvement of animal welfare, defined as the prevention of suffering and increasing the presence of positive feelings, or comfort is an important factor in livestock management (Broom, 1992. ibid). Measurements of impaired biological functioning, particularly those connected to decreased health and increased physiological stress responses, are used to evaluate the welfare status of farm animals.
There is an unmet need for an efficient, safe treatment for reducing the dry period in dairy herd without negatively affecting the milk yield, for increasing milk yield and hygiene and for keeping and/or improving the welfare of livestock animals. Furthermore, it would be highly advantageous to have pharmaceutical compositions in the form of clear, ready to use solution, comprising casein-derived peptides.